Spurious reflections and scattering centers other than gold will produce unwanted bright spots in the images. Even thresholding the image taken at the resonance peak (532 nm) will result in many false positive regions.
The presence of a plasmon resonance in the scattering spectrum of gold nanoparticles was used as a signature to distinguish them.
The ratio between the scattering cross section at 532 nm and 473 nm was found to be larger than 1.4 for gold monomers [Fig. 2(a), solid thin line] using Mie theory [1]} and even larger for dimers (dashed line) as calculated using GMMie, a multiparticle extension of the Mie theory [2]}, [3]}.
In contrast, non metallic scattering centers lack of a plasmon resonance and therefore yield a smaller ratio between 532 nm and 473 nm \(C_{sca}\)  (solid thick line).
Therefore, by imaging at these two wavelengths and thresholding the ratio image above 1.4, the pixels containing gold nanoparticles were further segmented.
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